Infection with human papillomaviruses (HPVs) is directly linked to the development of anogenital neoplasia. HPVs encode two oncoproteins, E6 and E7, that disrupt cell cycle checkpoints to allow viral replication to occur in cells that are normally quiescent. The mechanism by which E7 bypasses G1 growth arrest signals is complex. The hypothesis that G1 bypass-deficient mutations in CR1 and the C-terminus of E7 fail to disrupt RB:E2F:HDAC complexes, and may also involve direct inactivation of the CDK inhibitor (CKI) p2iCIP1 will be examined. Mutated E7 proteins will be assayed for associations with E2F, histone deacetylase activity and p21, and the results will be correlated with the ability to bypass DNA damage or a p21 C-terminal peptide induced arrest. Senescence is another form of G1 arrest that is controlled by the accumulation of CKIs and the shortening of telomeres. Immortalization of epithelial cells required inactivation of the p16INK4A/RB pathway but apparently not p53/ARF, leading to the hypothesis that the CKIs that regulate senescence may be cell-type specific. The senescence related expression of INK4A and ARF will be examined, as will their role in fibroblast immortalization. Additionally, new binding partners of ARF will be characterized for their ability to affect transcriptional activation or growth arrest. The discovery that E6 induces telomerase in epithelial cells links the viral oncoprotein with both blocks to senescence. We hypothesize that the ability of E6 to activate transcription of hTERT is linked to binding to the ubiquitin ligase, E6AP. New proteins that associate with the E6/E6AP complex with be characterized for their ability to affect telomerase. The relationship of these proteins to pathways that result in phosphorylation of c-MYC will be explored. Taken together. Taken together these experiments will contribute to an understanding of how E6 and E7 deregulate the controls that normally regulate G1 arrest.